The basic helix-loop-helix (bHLH) family of transcriptions factors is a structurally complex and functionally heterogeneous group in which over 400 bHLH-domain containing proteins have been identified. bHLH proteins are known to be involved with myriad cellular processes including neurogenesis, myogenesis, cell proliferation, cell fate determination, and tissue differentiation among other essential developmental processes.
The highly conserved bHLH domain that characterizes members of this family consists of two amphipathic helixes separated by a loop that mediates homo- and/or hetero-dimerization (Ledent, V., O. Paquet, and M. Vervoort, Genome Biol 2002; 3: RESEARCH0030). The basic (b) component of the bHLH domain includes a short component of mainly basic residues that bind to a consensus DNA sequence element, the “E-box” (CANNTG). The helix-loop-helix (HLH) component of the bHLH proteins is the structural mediator of the sequestering process and comprises a highly hydrophobic oligomerization region of approximately 50 residues which allows the formation of homodimeric or heterodimeric complexes between different family members. The relative placement of the bHLH domain in a particular protein can vary significantly. For example, the bHLH domain can be located at the COOH end of the protein (as in the Myc proteins), at the NH end (as in Sim), or in an intermediate position (MyoD).
Inhibitor of differentiation (Id) genes encode members of the helix loop helix (HLH) family of transcription factors that inhibit transcription by forming inactive heterodimers with bHLH proteins (Benezra, R., et al., Cell 1990; 61: 49-59). Typically, bHLH proteins form heterodimers with other bHLH proteins and their basic domain binds to the E box, activating transcription. Id proteins lack the basic domain necessary for DNA binding and act primarily as dominant-negative regulators of bHLH transcription factors by sequestering and/or preventing DNA binding of ubiquitously expressed (e.g., E12, E47, E2-2) or cell-type-restricted (e.g., Tal-1, MyoD) factors.
While Id proteins generally act as negative regulators of differentiation, depending on the specific cell lineage and developmental stage of the cell, Id proteins can also act as positive regulators. (Lasorella, A., et al., Cancer Res, 2002. 62(1): p. 301-6) Because bHLH proteins are mainly involved in the regulation of the expression of tissue specific and cell cycle related genes, Id-mediated sequestration or repression of bHLH proteins serves to block differentiation and to promote cell cycle activation. In general, expression of Id mRNA is highest in proliferating cells including carcinomas and low or usually absent in quiescent or terminally differentiated cells. (Coppe, J. P., A. P. Smith, and P. Y. Desprez, Exp Cell Res, 2003. 285(1): p. 131-45; Sikder, H. A., et al., Cancer Cell, 2003. 3(6): p. 525-30; de Candia, P., R. Benezra, and D. B. Solit, Adv Cancer Res, 2004. 92: p. 81-94; Fong, S., R. J. Debs, and P. Y. Desprez, Trends Mol Med, 2004. 10(8): p. 387-92; Wong, Y. C., X. Wang, and M. T. Ling, Apoptosis, 2004. 9(3): p. 279-89; Ruzinova, M. B. and R. Benezra, Trends Cell Biol, 2003. 13(8): p. 410-8)
The four known Id proteins (Id1, Id2, Id3 and Id4) have highly conserved HLH domains, but divergent N- and C-terminal domains. In addition to modulating bHLH proteins, they have also been shown to modulate the activity of non-bHLH proteins such as retinoblastomas, MIDA1, ETS-domain transcription factors, Pax2, Pax5, Pax8 and ELK-1.
Discovery of molecules capable of inhibiting Id proteins has been difficult due to the location of the HLH-bHLH interactions in the nucleus and the relatively delocalized nature of the protein-protein interactions underlying the interaction. Additionally, the high degree of homology between HLH and bHLH domains makes the isolation of anti-Id molecules that are not also Ids challenging. Inhibiting Id expression through antisense or siRNA has been considered (Henke E, et al., Nat. Biotechnol., 2008. 26(1): p 91-100.) however these approaches are not desirable for commercialization and widespread use. There is therefore a need for additional means for inhibiting Id expression.